The ability to strictly regulate apoptosis is particularly important for postmitotic cells such as neurons, cardiomyocytes and myotubes because these cells have limited regenerative potential and are maintained for the lifetime of the organism. Failure to restrict apoptosis can result in increased vulnerability to cell death, as seen in many degenerative diseases. Therefore, an understanding of survival mechanisms in postmitotic cells is of physiological and pathological importance. The mitochondrial release of cytochrome c (cyt c) is a crucial event that triggers caspase activation during apoptosis. Here, we have identified a novel mechanism for regulating cyt c-mediated apoptosis engaged by neurons and other postmitotic cells, where cytosolic cyt c itself is targeted for proteasomal degradation. Importantly, while we do not observe cyt c degradation in primary mitotic cells such as fibroblasts, we find mitochondrial-released cyt c to be also degraded in certain cancer cells. As evasion of apoptosis is an important feature of both postmitotic and cancer cells, targeting cytosolic cyt c for degradation could be a shared mechanism used by these cells for survival. In this proposal, our goals are to identify the molecular mechanisms by which cytosolic cyt c is targeted for degradation. In Aim 1, we will define the factors which determine whether or not a cell will target cytosolic cyt c for degradation, including examining whether low levels of Apaf-1 in cells is a key determinant for cyt c degradation. In Aim 2, we will examine cyt c ubiquitination and identify the specific residues of cyt c that are targeted for ubiquitination. Our hypothesis is that ubiquitinated cyt c is not able to bind Apaf-1. We will test this, and examine whether conditions that block cyt c degradation increase the vulnerability of postmitotic cells to apoptosis. Our focus in Aim 3 is to identify the E3 ubiquitin ligase that targets cyt c for degradation. Our preliminary results suggest that cyt c binds to Hsp70 and the E3 Ligase CHIP (Carboxyl Heat shock protein 70-Interacting Protein) in the cytosol. Therefore, we will test whether the ubiquitination and degradation of cytosolic cyt c is mediated by CHIP in postmitotic and cancer cells. PUBLIC HEALTH RELEVANCE: Our plans here are to investigate how the cell death pathway is regulated in mammalian cells. We have discovered a novel mechanism engaged by postmitotic cells such as neurons, cardiomyocytes and myotubes that highly restricts cell death and likely ensures their long term survival. Understanding the survival mechanisms used by postmitotic cells has enormous clinical significance because increased death of these cells is central to the pathology of many neurodegenerative diseases, cardiac pathologies and muscular dystrophies.